The long term objective of this study is to understand the nature of bone cell calcium regulation. The endogenous and exogenous factors that control this parameter will be assessed and the potential interrelationships between these factors and calcium in the regulation of bone metabolism will be evaluated. The effects of parathyroid hormone (PTH), prostaglandin E2 (PGE2), epidermal growth factor (EGF) and interleukin -I(IL-l) will be studied in normal rat osteoblastic cells and the osteosarcoma lines - UMR-106 and SaOS-2. The interrelationships between putative effects of these agents on calcium and phosphoinositide metabolism will also be studied by investigating the regulation of this phospholipid in the bone cells and by studying the effects of inositol phosphates on calcium mobilization in these isolated cell systems. Interrelationships between arachidonic and cytosolic calcium will also be evaluated and the effects of arachidonic acid on intracellular calcium mobilization will also be studied. The role of protein kinase C in the regulation of osteoblastic calcium metabolism will also be studied using inhibitors and activators of this enzyme. Studies on protein, DNA synthesis and plasminogen activator will also be conducted in an attempt to make correlations with changes in cytosolic calcium. Arachidonic acid metabolism will also be studied in the various cell systems to better understand the interrelationship between cyclooxygenase products and cytosolic calcium. Cytosolic calcium levels will be measured using the Fura 2 fluorescent assay with cells in suspension and attached to coverslips. Phosphoinositol metabolism will be studied with thin layer and ion exchange chromatography and high performance liquid chromatography. Arachidonic acid metabolism will also be studied by high performance liquid chromatography. These studies should lead to a better understanding of the nature of bone cell metabolism and aid in the development of therapy for systemic and localized bone metabolic disorders.